VILNIUS UNIVERSITY INSTITUTE OF BIOTECHNOLOGY ...

VILNIUS UNIVERSITYINSTITUTE OF BIOTECHNOLOGYGražvydas LukinavičiusSEQUENCE-SPECIFIC LABELING OF DNA VIA METHYLTRANSFERASE-DIRECTED TRANSFER OF ACTIVATED GROUPS (MTAG)Summary of doctoral dissertationPhysical science, biochemistry (04 P), proteins, enzymology (P 310)Vilnius, 2007

The work presented in this doctoral dissertation has been carried out at the Institute of Biotechnologyfrom 2002 to 2006.Dissertation is maintained by externScientific consultant:Prof. Dr. habil. Saulius Klimašauskas (Institute of Biotechnology, physical sciences,biochemistry - 04 P, proteins, enzymology - P310)Evaluation board of dissertation of Biochemistry trend:Chairman:Members:Official opponents:Prof. Dr. habil. Kęstutis Sasnauskas (Institute of Biotechnology, physical sciences,biochemistry - 04 P, proteins, enzymology - P310)Prof. Dr. habil. Vida Kirvelienė (Vilnius University, physical sciences,biochemistry - 04 P,nucleic acids, protein synthesis - P320)Prof. Dr. habil. Eugenijus Butkus (Science Council of Lithuania, physical sciences, chemistry- 03 P, organic chemistry - P390)Dr. Rolandas Meškys (Institute of Biochemistry, physical sciences, biochemistry -04 P,proteins, enzymology - P310)Prof. Dr. habil. Virginijus Šikšnys (Institute of Biotechnology, physical sciences, biochemistry– 04 P, proteins, enzymology - P310)Prof. Dr. habil. Gervydas Dienys (Institute of Biotechnology, physical sciences, chemistry -03 P, organic chemistry - P390)Dr. habil. Narimantas Čėnas (Institute of Biochemistry, physical sciences, biochemistry - 04P, proteins, enzymology - P310)The thesis defense will take place at 1 p. m. on 12 th of October, 2007, at the Institute of Biotechnology.Address: Graičiūno 8, LT-02241, Vilnius, Lithuania.The abstract of the thesis was distributed on 3 rd of September, 2007.The thesis is available at the Library of Institute of Biotechnology and at the Library of VilniusUniversity.2

VILNIAUS UNIVERSITETASBIOTECHNOLOGIJOS INSTITUTASGražvydas LukinavičiusMETILTRANSFERAZIŲ KATALIZUOJAMAS FUNKCINIŲ GRUPIŲ ĮVEDIMASSPECIFINĖSE DNR SEKOSEDaktaro disertacijaFiziniai mokslai, biochemija (04 P), baltymai, enzimologija (P310)VILNIUS, 20073

Disertacija rengta 2002-2006 metais Biotechnologijos Institute.Disertacija ginama eksternu.Mokslinis konsultantas:prof. habil. dr. Saulius Klimašauskas (Biotechnologijos Institutas, fiziniai mokslai, biochemija –04 P, baltymai, enzimologija – P310)Disertacija ginama Vilniaus Universiteto Biochemijos mokslo krypties taryboje:Pirmininkas:prof. habil. dr. Kęstutis Sasnauskas (Biotechnologijos institutas, fiziniai mokslai, biochemija -04 P, baltymai, enzimologija - P310)Nariai:prof. habil. dr. Vida Kirvelienė (Vilniaus universitetas, fiziniai mokslai, biochemija - 04 P,nukleorūgštys, baltymų sintezė - P320)prof. habil. dr. Eugenijus Butkus (Lietuvos Mokslo taryba, fiziniai mokslai, chemija -03 P,organinė chemija - P390)dr. Rolandas Meškys (Biochemijos institutas, fiziniai mokslai, biochemija – 04 P, baltymai,enzimologija - P310)prof. habil. dr. Virginijus Šikšnys (Biotechnologijos institutas, fiziniai mokslai,biochemija - 04 P, baltymai, enzimologija - P310)Oponentai:prof. habil. dr. Gervydas Dienys (Biotechnologijos institutas, fiziniai mokslai, chemija - 03 P,organinė chemija - P390)habil. dr. Narimantas Čėnas (Biochemijos institutas, fiziniai mokslai, biochemija - 04 P,baltymai, enzimologija - P310)Disertacija bus ginama viešame Biochemijos mokslo krypties tarybos posėdyje 2007 m. spalio 12 d.13 val. Biotechnologijos Instituto konferencijų salėjeAdresas: Graičiūno 8, LT – 02241, Vilnius, LietuvaDisertacijos santrauka išsiuntinėta 2007 m. rugsėjo 3 d.Disertaciją galima peržiūrėti Biotechnologijos Instituto ir Vilniaus Universiteto bibliotekose4

CONTENTSCONTENTS ......................................................................................................................................................... 5INTRODUCTION................................................................................................................................................ 6MATERIALS AND METHODS......................................................................................................................... 8RESULTS AND DISCUSSION ........................................................................................................................ 131 Design of novel activated AdoMet analogs........................................................................................... 132 Synthesis and purification of AdoMet analogs...................................................................................... 132.1 Synthesis of model AdoMet analogs ......................................................................................... 142.2 Synthesis of cofactor analogs bearing a primary amino group ................................................. 152.2.1 Synthesis of N-Boc-protected aminoalcohols ........................................................................... 152.2.2 Activation of N-Boc-protected aminoalcohols, coupling to AdoHcy and N-Boc removal...... 162.3 Purification and structure elucidation........................................................................................ 163 Chemical stability of the AdoMet analogs ............................................................................................ 194 Activity of AdoMet analogs in enzymatic reactions .............................................................................214.1 Activity with wild type cytosine DNA methyltransferases....................................................... 214.2 Engineering the cofactor binding pocket in HhaI methyltransferase........................................ 225 Composition analysis of transalkylated DNA ....................................................................................... 246 Sequence specific blockage of protein-DNA interaction ...................................................................... 277 Targeted labeling of DNA by methyltransferase-directed Transfer of Activated Groups (mTAG)..... 288 Discussion .............................................................................................................................................. 31CONCLUSIONS................................................................................................................................................ 34LIST OF PUBLICATIONS ............................................................................................................................... 35ACKNOWLEDGEMENTS ............................................................................................................................... 36CURRICULUM VITAE .................................................................................................................................... 36REZIUMĖ .......................................................................................................................................................... 37REFERENCES................................................................................................................................................... 385

INTRODUCTIONLiving organisms are dynamic systems of interacting biopolymers and their metabolites.Functional analysis of such systems often stands on the ability to specifically visualize selectedbiomolecules. To minimize possible artifacts, a small reporter group is to be attached to a selectedspecific position on a biomolecule under investigation. Several approaches for sequence-specific noncovalentlabeling of DNA have been proposed to date. However these techniques suffer from a majorproblem – the lack of tight coupling between a reporter moiety and its biological target, leading to highbackground and low detection sensitivity (Dirks et al., 2006; Ghosh et al., 2006). On the other hand,examples of sequence-specific covalent modification of DNA can be found in nature. The mostabundant covalent modification of DNA is methylation of nucleobases. It serves to expand theinformation content of the genome in organisms from bacteria to humans, and, for example, is part ofan intricate epigenetic regulatory network in higher vertebrates (Doerfler, 2006). The DNA methylationreactions are catalyzed by DNA methyltransferases – a class of enzymes that transfer a methyl groupfrom the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto predefined target sites on DNA.Despite the high importance of transmethylation reactions in biology, the transferred methyl group haslimited utility for practical applications. However, the ability of most MTases to catalyze highly specificcovalent modifications of biopolymers makes them attractive molecular tools, provided that transfer oflarger chemical entities can be achieved.Several attempts to achieve methyltransferase-directed transfer of extended groups from analogsof AdoMet have been reported. Early studies of AdoMet analogs (Parks, 1958; Schlenk et al., 1975) inwhich the transferable group on the sulfonium center is replaced by an extended aliphatic chainindicated that relatively short chemical groups, such as ethyl and propyl, can be transferred byMTases, but the transfer rates decline drastically as the size of the transferable group increases(methyl >> ethyl > propyl). This effect largely derives from destabilizing steric interactions in theextended aliphatic groups during nucleophilic substitution (S N 2) reactions. These early experimentsthus did not show much promise for targeted labeling of biomolecules using such extended AdoMetanalogs. The first success in covalent labeling of specific DNA sequences was achieved using N-aziridine cofactor mimics, which make use of a nucleophilic ring-opening reaction leading tomethyltransferase-directed coupling of the whole cofactor to DNA. By attaching chemical groups to theaziridine cofactors, they have been shown to work in combination with DNA MTases as deliverysystems for various functional or reporter groups. However, an inherent feature of this system is thatcovalent coupling of the two substrates leads to potent product inhibitors preventing further turnovers.Therefore, the DNA MTases have to be used in stoichiometric amounts with respect to their targetsites, and their tight association with the DNA may interfere with downstream applications (Pljevaljcicet al., 2004; Comstock et al., 2005; Klimasauskas et al., 2007).The above described problems prompted us to revisit the chemistry of sulfonium AdoMetanalogs. It is known that allylic and propargylic compounds are highly reactive in S N 2 reactions,suggesting that the rate of a methyltransferase-directed reaction can be enhanced by placingunsaturated bonds next to the transferable carbon in the extended side chains of AdoMet analogs.The goal of this work was to explore the potential of the propargylic system in transalkylationreactions catalyzed by AdoMet-dependent DNA methyltransferases. A series of AdoMet analogs withextended propargylic side chains have been chemically synthesized and examined in enzymaticreactions with all three types of DNA methyltransferases. The efficiency of transalkylation has beenfurther enhanced by rational engineering of the cofactor binding center in a model DNA cytosine-5methyltransferase, M.HhaI. This enzyme is a small protein that has been thoroughly characterized withrespect to the catalytic mechanism, interaction with cofactor and DNA (Wu et al., 1987; Cheng et al.,1993; Vilkaitis et al., 2001; Merkiene et al., 2005; Neely et al., 2005; Estabrook et al., 2006). Based onthe demonstrated methyltransferase-directed transfer of extended groups, a novel strategy forsequence-specific functionalization and labeling of DNA has been proposed.6

Specific aims:1. To chemically synthesize a series of activated AdoMet analogs with extended propargylic sidechains replacing the methyl group.2. To investigate chemical stability of the extended AdoMet analogs and assess their activity in DNAmethyltransferase-catalyzed transalkylation reactions.3. To engineer the cofactor binding center in the HhaI MTase for efficient transfer of extendedgroups from the AdoMet analogs.4. To demonstrate sequence-specific functionalization and labeling of natural DNA molecules usingthe AdoMet analogs and engineered DNA methyltransferases.Scientific novelty:The specificity of an enzymatic reaction has been successfully redesigned via syntheticmodification the AdoMet cofactor and rational engineering of the cofactor binding center in a DNAcytosine-5 methyltransferase. It was shown for the first time that sterically-impaired linear side chainscan be activated for enzymatic S N 2 reactions by introducing unsaturated carbon-carbon bonds next tothe transferable carbon atom. We also demonstrate for the first time an efficient site-specific transfer oflinear carbon chains containing primary aliphatic amino groups, followed by amino-specific covalentcoupling of reporter groups onto DNA.Practical value:A synthetic strategy has been developed for obtaining novel analogs of the ubiquitous cofactor ofAdoMet-dependent methyltransferases that contain functional or reporter groups within extendedtransferable side chains. The newly developed mTAG technology (methyltransferase-directed Transferof Activated Groups) was shown to be effective for sequence-specific functionalization and labeling ofplasmid DNA, which can be further applied for analysis of genomic DNA methylation. These findingspave the way for targeted labeling of other biopolymers such as RNA and proteins using a myriad ofhighly specific AdoMet-dependent methyltransferases known in nature. These new molecular toolsenvision numerous potential applications ranging from probes for genetic screening technologies tomolecular building blocks in DNA-based nanobiotechnology.Findings presented for defense:1. S-Adenosyl-L-methionine (AdoMet) analogs with extended propargylic side chains have beenobtained by direct chemoselective S-alkylation of S-adenosyl-L-homocysteine.2. AdoMet analogs bearing unsaturated bonds next to the reactive carbon are more active in S N 2and A N reactions than corresponding saturated compounds.3. Adenine-N6, cytosine-N4 and cytosine-C5 DNA methyltransferases catalyze the transfer ofextended groups onto DNA with full retention of their target specificity.4. Steric engineering of the cofactor binding center in a DNA cytosine-C5 methyltransferase,M.HhaI, leads to an enhanced transfer of extended side chains from synthetic AdoMet analogs.5. AdoMet analogs containing a primary aliphatic amino group in the extended side chain can beused for methyltransferase-directed sequence-specific labeling of natural DNA.7

MATERIALS AND METHODSChemicals and enzymesAll reagents used in this study were reagent-grade commercial products. Gradient-grade HPLCsolvents were purchased from Roth. Dynabeads® M-280 Streptavidin-coated magnetic beads wereobtained from Invitrogen. Ethanol was obtained from AB Vilniaus degtinė. Nuclease P1 was purchasedfrom Roche, bovine serum albumin (BSA) was obtained from Pierce Biotechnology, all other enzymes,their reaction buffers and kits were purchased from Fermentas UAB and used according to themanufacturers' recommendations.Methyltransferases:WT M.BcnIB prepared by dr. E. Merkienė.WT M.BfiC2 and WT M.Eco31I prepared by G. Urbanavičiūtė.M.HhaI variant N304A prepared by Z. Staševskij and J. Šlyžiūtė.WT M.TaqI was a kind gift from prof. E. Weinhold.OligonucleotideshhaIM gene sequencing and PCR primers:H25‘-GCTGAGTGCGTTTATTCTAAT-3‘H55’-TTTTCGCAATGATCTCAATATTC-3‘V15’-CTTAGATTCAATTGTGAGCGG-3’V25’-ATCAACAGGAGTCCAAGCTCAGC-3’T7PP5‘-TAATACGACTCACTATAGGG-3‘Oligonucleotides for fluorescence titrations and LC/MS analysis M.HhaI recognition site isboldface. M.BcnIB recognition site is underlined. M stands for 5-methylcytosine and P – for2-aminopurine:GPGC:GMGC:Uni#1.1Uni#1.25’-TACAGTATCAGGPGCTGACCCACAA-3’,3’-TGTCATAGTCCGMGACTGGGTGTTG-5’,5‘-ATAGCCAGGACCCGGCGCGTAATG-3‘3’-TATCGGTCCTGGGCCGCGCATTAC-5’All oligonucleotides were obtained from MWG-Biotech AG (HPSF grade). DNA duplexes wereproduced by annealing appropriate oligonucleotides in a thermal cycler (85°C 5 min.; 85°C→4°C,cooling speed 0.01°C/min.).8HPLC columns:Analytical: Discovery HS C18 75x2.1 mm, particle size 3 µm (Supelco) and Discovery C18150x2.1 mm, particle size 5 µm (Supelco)Preparative: Discovery C18 250x10 mm, particle size 5 µm (Supelco) and Discovery HS C18150x10 mm, particle size 5 µm (Supelco)Chemical synthesis of AdoMet analogsPreparation of aminoalcoholsA solution (150 ml) of 4-phthalimidobut-2-yn-1-ol (1 equiv., 7.6 g, prepared from 2-butyn-1.4-diolaccording to (Thomson et al., 2003)) or 6-phthalimidohex-2-yn-1-ol (1 equiv., 8.5 g, prepared from 5-

chloro-1-pentyne according to (Brandsma et al., 1981) and (Sheehan et al., 1950)) in methanol wastreated with hydrazine hydrate (2 equiv., 3.46 ml). The reaction mixture was heated with reflux for 2 hand after cooling to room temperature the solvent was removed under reduced pressure (100 mmHg,40°C). Water and ethanol (100 ml, 1:1 mixture) and conc. hydrochloric acid (100 ml) were added tothe residue. The mixture was heated with reflux for 20 min and the precipitate removed by filtration.The filtrate was concentrated under reduced pressure (10 mmHg, 60°C). The resulting 4-aminobut-2-yn-1-ol hydrochloride residue was crystallized from methanol as a white solid. The 6-aminohex-2-yn-1-ol was used in further reactions without crystallization.Extending aminoalcohols by CDI coupling4-[(tert.-butoxycarbonyl)amino]butanoic acid (1 equiv., 5 g, prepared in analogy to (Greene et al.,1999)) was dissolved in anhydrous tetrahydrofuran (20 ml), carbonyldiimidazole (CDI) (1.1 equiv., 4.56g) was added, and the resulting clear solution was stirred at room temperature for 2 h. Then, theaminoalcohol (1 equiv.) and trietylamine (2 equiv., 7 ml) were added and stirring was continued atroom temperature for 2 h. The solvent was removed under reduced pressure (50 mmHg, 40°C) andthe crude product was purified by column chromatography (silica gel, 40 g, chloroform/ethylacetate1:1). Product containing fractions were pooled and solvent was removed under reduced pressure.Activation of alcohols by sulfonylationThe reaction was performed following the previously described procedure (Roberts, 1971). 4-Nitrobenzenesulfonyl chloride (1.1 equiv., 0.90 g) and sodium hydroxide (5 equiv., 0.74 g) were addedto a solution of protected aminoalcohol (1 equiv.) in methylene chloride (15 ml) at 0°C. After stirringthe reaction mixture for 3 h at room temperature sodium hydroxide was filtered, the reaction wasquenched with 20 ml of cold water, extracted with methylene chloride (3x10 ml) and the combinedorganic layers dried over sodium sulfate. The sample was passed through a glass filter andconcentrated under reduced pressure (200 mmHg, 30°C) as a slightly yellow solid.S-Alkylation of S-adenosyl-L-homocysteineAn activated alcohol (100–200 equivalents of triflate, prepared according to (Ross et al., 2000) or10-30 equivalents of 4-nitrobenzenesulfonyl ester (see above)) was slowly added to S-adenosyl-Lhomocysteine(AdoHcy) (1 equiv., 10–20 mg) in a 1:1 mixture of formic acid and acetic acid (0.5–1.0ml) at 0°C. The solutions were allowed to warm up to room temperature and incubated with shaking.After specified times (2-8 h) the reactions were quenched by adding water (5–10 ml). The aqueousphase was extracted three times with equal volume of diethyl ether and water was removed in rotaryevaporator (10 mmHg, 30°C). Residue was dissolved in 10 ml of HPLC buffer. Purification of AdoButinand AdoPentin was performed by preparative reversed-phase HPLC. The 4-nitrobenzenesulfonatewas removed by passing solution through Dowex 1 anion exchanger column prior the HPLCpurification.Purification of AdoMet analogsPurification of AdoMet analogs was performed by preparative reversed-phase HPLC eluting witha linear gradient of two solvents: A (20 mM HCOONH 4 , pH 3.5) and B (80% methanol solution inwater) at a flow rate of 4.5 ml/min. For details see Table I.9

Table I. AdoMet analogs purification conditions.AdoMetanalogUsedcolumnGradientparametersAdoButin andAdoPentinAdoButinNH 2(protected)AdoButinGABANH 2 AdoHeksinGABANH 2Discovery C18 250x10 mmDiscovery HS C18150x10 mmTime,min.B solvent,%Time,min.B solvent,%Time,min.B solvent,%Time,min.B solvent,%0 10 0 10 0 08 50 2 10 7 09 100 10 100 Isocratic9 100014 100 15 100 conditions12 10015 10 16 10 14 025 10 35 1035 0Compounds were detected by their absorption at 260 and 280 nm. Enriched Fractions werepooled, lyophilized under reduced pressure in a rotary evaporator (10 mmHg, 30°C) and desalted bypassing through reverse phase C-18 silica gel. Structure of novel compound was confirmed by NMRmeasurements and yields determined by UV absorption of the adenine chromophore (ε 260 = 15400 lmol -1 cm -1 ).NMR measurements1H NMR spectra were recorded at 300 MHz and 13 C NMR at 75 MHz on a Varian Unity Inova 300spectrometer in CDCl 3 or D 2 O and are reported in δ ppm (multiplicity, coupling constant (J) in hertz(Hz), integrated intensity and assigned group) relative to TMS, using the residual solvent peaks asinternal standards.Engineering of mutants of M.HhaIPlasmids containing hhaIM gene with single Q82A and N304A mutations had been previouslyproduced as derivatives in expression vectors pHH5.3 or pETHH2111, respectively, and kindlyprovided by E. Merkienė and Z. Staševskij. pTZ-HE contained the Y254S mutation in a truncatedMTase gene. Full length hhaIM gene was restored by cloning R.Acc65I-R.Eco91I fragment to theexpression vector pHH5.3. M.HhaI variants containing two or three mutations were constructed byrecombining appropriate fragments with single mutations. For this purpose, unique sites for R.Eco81I,R.Rco88I, R.Eco91I, Acc65I and R.HindIII were used. Recombinant plasmids were transformed intothe E. coli ER2267 strain. Transformants containing appropriate diagnostic sites were selected. Allmutations were confirmed by complete sequencing of the genes. For protein expression, pHH5.3 andpETHH2111 vector based plasmids were transformed into the E. coli ER2267 and ER2566 strains,respectively.Protein expression and purificationM.HhaI and its variants were expressed in E. coli ER2267 or ER2566 cells containing thepHH553 or pETHH2111 respectively ((Daujotyte et al., 2003) and G. Vilkaitis, unpublished data).Protein expression was induced by adding IPTG to the 0.4 mM final concentration. MTases wereselectively enriched by exploiting a high salt (0.4 M NaCl) back-extraction from the cell debris.Following extensive dialysis to remove bound endogenous AdoMet, MTases were purified by passingthrough a pre-column of Q-Sepharose followed by column chromatography on S-Sepharose (Holz etal., 1998). All proteins appeared as sole bands (> 95%) in Coomassie-stained polyacrylamide gels.Protein concentrations were estimated using a Coomassie G-250 assay with BSA as standard andfurther refined by active site titration with a 25-mer fluorescent duplex GPGC/GMGC. The molecularmass of purified enzymes was verified by nebulizing samples using electrospray followed by mass tocharge ratio measurements in a single quadruple mass spectrometer (Hewlett-Packard 1100 seriessystem).10

DNA protection assay for alkyltransferase activityActivity of AdoMet analogs in the enzymatic reactions catalyzed by M.BcnIB or M.HhaI and itsvariants was tested on phage lambda DNA (215 M.HhaI recognition sites and 114 M.BcnIB recognitionsites). Analogous tests with M.Eco31I and M.BfiIC2 were performed on plasmid pUC19 DNA (1recognition site of M.Eco31I and 2 recognition sites of M.BfiC2).Twofold serial dilutions (15 µl) of DNA MTases starting with a stoichiometric ratio of DNA MTasesto recognition sites in buffer (M.HhaI/M.BcnIB buffer: 50 mM Tris-HCl, 10 mM NaCl, 0.5 mM EDTA, 2mM 2-mercaptoethanol, 0.2 mg/ml BSA, pH 7.4; M.Eco31I buffer: 20 mM MOPS, 20 mM Tris, 20 mMCAPS, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 10.0; M.BfiIC2 buffer: 20 mM MOPS, 20 mM Tris, 20 mMCAPS, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 9.0) containing phage lambda or plasmid pUC19 DNA andAdoMet analogs (total concentration of 300 µM) were incubated at 37°C (M.HhaI, M.BcnIB andM.Eco31I) or 30°C (M.BfiIC2) for 1 or 4 h. The reaction was stopped by heating at 80°C for 10 min. Incase of M.Eco31I and M.BfiIC2 pH of the samples was adjusted to 7.5 and 8.0 respectively by adding0.1 M HCl prior to restriction digestion. Afterwards a solution of restriction endonucleases (R.Hin6I toM.HhaI incubation, R.BcnI – M.BcnIB, R.Eco31I – M.Eco31I, R.BfiI – M.BfiC2, 2-10 u/1 µg DNA) andMgCl 2 (final concentration of 10 mM) was added to each sample and incubation was continued at37°C for 3 h. Reaction was stopped by adding proteinase K (0.2 mg/ml), SDS (0.1%) and heating at55°C for 30 min. Samples were supplemented with 1/6 volume of orange loading dye (0.2% orange G,0.05% xylene cyanol FF, 60% glycerol, 60 mM EDTA) and analyzed by agarose gel (1%)electrophoresis.Composition analysis of transalkylated DNAEnzymatic modifications with M.HhaI (Q82A or Q82A/N304A variants) and M.BcnIB (WT) wereperformed by incubation of an oligonucleotide duplex Uni#1.1:Uni#1.2 (10 µM) with AdoMet analogs(final concentration of 300 µM) and MTase (12.5 µM) in M.HhaI/M.BcnIB buffer (50 mM Tris-HCl, 10mM NaCl, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, 0.2 mg/ml BSA, pH 7.4) at 37°C for 2-4 h.Samples were then incubated at 80°C for 10 min and treated with Proteinase K (0.2 mg/ml) and SDS(0.1%) at 55°C for 1 h. The modified oligodeoxynucleotides were desalted by gel filtration using G-25columns. Afterwards samples were supplemented with P1 buffer (10 mM Tris-HCl, 10 mM MgCl 2 , 1mM Zn(OAc) 2 , pH 7.5) containing Nuclease P1 (1.5 u) and calf intestine alkaline phosphatase (30 u)and incubated at 42°C for 4 h. Obtained hydrolysis solution was passed through a Microcon YM-3 spincolumn and subjected to LC/MS analysis.LC/MS analysisNucleosides or cofactor decomposition products were analyzed by reversed-phase HPLCcoupledelectrospray ionization mass spectrometry (Hewlett-Packard 1100 series system). Sampleswere loaded onto a column and eluted with linear gradient of two solvents A (20 mM HCOONH 4 , pH3.5) and B (80% methanol solution in water) at a flow of 0.3 ml/min (see table II). Separatedcompounds were detected by an in-line diode array UV absorbance detector at 210, 260 and 280 nm.Additionally, UV absorbance spectra were acquired (190-400 nm wavelength interval) at peak maximaand solvent contributions were removed by subtracting background spectra recorded before and afterthe peaks. For the mass spectrometric analysis of nucleosides post-column equal co-flow of 96%methanol, 4% formic acid and 1 mM sodium formate was used. For the MS detection of AdoMet, itsanalogs and decomposition products a post-column equal co-flow of 99% methanol, 1% CF 3 COOHwas used. Mass spectra were acquired in 50-800 m/z range in positive ion mode. Ionization capillaryvoltage was 5000 V, fragmentor voltage was in 100-120 V interval, drying gas temperature was 300-350°C and flow rate was 10-12 l/min.11

Table II. HPLC separation conditions.AnalyzedcompoundsNucleosidesAdoMet and its analogscolumnused Discovery C18 150x2.1 mm Discovery HS C18 75x2.1 mm Discovery HS C18 75x2.1 mmColumntemperatureGradientparametersTime,min.45°C 30°C 20°CB solvent,%Time,min.B solvent,%Time,min.0 0 0 0 0 05 0 3 0 2 020 70 18 25 7 10021 100 20 100 10 10026 100 25 100 11 027 0 27 0 20 0B solvent,%40 0 40 0 - -MTase-directed amino-modification of plasmid DNAReaction solutions containing equimolar amounts of DNA MTases and plasmid pBR322 DNA(WT M.TaqI: 0.5 µM; M.HhaI variant Q82A/N304A: 2.5 µM) in M.TaqI (20 mM Tris-HOAc, 50 mMKOAc, 10 mM Mg(OAc) 2 , 1 mM DTT, 0.01% Triton X-100, 0.1 mg/ml BSA, pH 7.9) or M.HhaI (50 mMTris-HCl, 10 mM NaCl, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, 0.2 mg/ml BSA, pH 7.4) reactionbuffer and AdoButinGABANH 2 (300 μM) were incubated at 60°C (M.TaqI) or 37°C (M.HhaI variantQ82A/N304A) for 1 h. Afterwards samples were diluted twice with water and extracted once withRoti®-Phenol (pH 8.0), two times with Roti®-Phenol/C/I and three times with chloroform. Aqueousphases were separated and DNA was precipitated by adding isopropanol (0.9 volume) and sodiumacetate (0.1 volume, 3 M, pH 7.0). Pellets were washed once with ice cold ethanol (75%) and dried.Modified DNA was dissolved in water to a final concentration 0.4–0.5 µg/µL as determined fromethidium stained agarose gels (0.8%).Labeling and analysis of MTase-modified plasmid DNAThe pBR322 DNA amino-modified with M.HhaI (variant Q82A/N304A) or with M.TaqI in thepresence of AdoButinGABANH 2 were treated with a Cy5-NHS or fluorescein-NHS(100-10000 equivalents) in 0.15 M sodium bicarbonate (pH 9.0) at room temperature for 1 h.Afterwards, DNA was precipitated by adding 0.9 volume of isopropanol and 0.1 volume 3 M sodiumacetate (pH 7.0). Ethanol-washed precipitate was dissolved in water and residual unattached dye wasremoved by passing solution through G-25 column. Approximately 1 µg of labeled DNA wasfragmented with R.GsuI (according to manufacturer’s recommendations) and analyzed by agarose gel(1.5%) electrophoresis. Gels were scanned with a Fuji FLA-5100 imaging system using a 473 nm(fluorescein labeled DNA) or 635 nm (Cy5 labeled DNA) laser. Densitometry of label distribution wasperformed using OptiQuant program version 3.The labeling of DNA with biotin-NHS (~10000 equivalents) was performed under the sameconditions. In this case M.TaqI amino-modified pBR322 or M.BseCI premethylated pBR322 werelabeled. Approximately 0.75 µg of labeled DNA was then fragmented with the R.FspAI and R.MbiI(according to manufacturer’s recommendations, reaction volume 30 µl). One half of a sample (15 µl)was treated with Dynabeads® M-280 Streptavidin-coated magnetic beads (200 µg, suspended in 5 µlrestriction enzyme buffer 66 mM Tris-acetate, pH 7.9, 20 mM magnesium acetate, 132 mM potassiumacetate and 0.2 mg/ml BSA) and incubated with shaking at 43°C for 2 h. Beads were removed with amagnet, washed twice with restriction enzyme buffer (30 µl) and resuspended in restriction enzymebuffer (15 µl). DNA fragments were recovered by extracting with an equal volume of Roti®-Phenol/C/Isolution. Finally, samples obtained after REase fragmentation, after treatment with streptavidin-coatedbeads and after elution from the beads were analyzed by agarose gel (1.2%) electrophoresis.12

RESULTS AND DISCUSSION1 Design of novel activated AdoMet analogsDNA methyltransferases naturally catalyze the transfer of the activated methyl group from thecofactor S-adenosyl-L-methionine (AdoMet) to adenine or cytosine within DNA leading to methylatedbases and the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy). The ability ofmethyltransferases to catalyze sequence-specific covalent modifications of DNA makes thempromising tools for biotechnology. Ideally, it would be desirable to transfer larger chemical entities withadditional functionalities to the target biomolecules. In principle, this can be achieved with AdoMetanalogs bearing larger transferable groups (Fig.1).AdoEt; R = -CH 2 -CH 3AdoProp; R = -CH 2 -CH 2 -CH 3RH 2 NS +CO 2 HNNONH 2NNAdoPropen; R = -CH 2 -CH=CH 2AdoPropin; R = -CH 2 -C CHAdoButin; R = -CH 2 -C C-CH 3HOOHAdoPentin; R = -CH 2 -C C-CH 2 -CH 3AdoButinNH 2 ;R=-CH 2 -C C-CH 2 -NH 2AdoButinGABANH 2 ;R=HNOHNONH 2NH 2AdoHeksinGABANH 2 ;R=Figure 1. Chemically synthesized AdoMet analogs used for enzymatic DNA alkylation reactions. AdoMetanalogs synthesized in prof. E. Weinhold laboratory are colored in red. Green colored are compounds synthesizedduring this study.Studies by Schlenk and Parks indicated that larger chemical groups like ethyl and propyl can betransferred from AdoEt (Parks, 1958) and AdoProp (Schlenk et al., 1975) by methyltransferases.However, it was also found that enzymatic alkyl transfer rates decline drastically with increasing size ofthe transferable group (methyl >> ethyl > n-propyl group).During the transition state of the methyl transfer (S N 2 reaction), the bond angles between thenucleophile as well as the leaving group and the three substituents at the reacting carbon are ~90°.This leads to destabilizing steric interactions if any of the three hydrogens are replaced by bulkierresidues. On the other hand, it is well known that the reaction rate can be rescued by placing a doublebond (allylic system) or triple bond (propargylic system) next to the reactive carbon. The chemicalrationale for this reactivation derives from conjugation (parallel alignment) of the p-orbital formed in thetransition state with the neighboring bonding or anti-bonding π-orbital (Dorwald, 2005).It was hypothesized that by placing an activating double or triple bond within the transferablechain next to the reactive carbon in the cofactor analogs, the MTase-catalyzed reaction rate will berescued by the above mentioned electronic effects. To test the hypothesis, a series of model AdoMetanalogs containing extended propargylic chains have been synthesized (Fig. 1).2 Synthesis and purification of AdoMet analogsNaturally AdoMet is formed by AdoMet synthetase (also called methionine adenosyltransferase,MAT) from adenosine triphosphate (ATP) and L-methionine (Takusagawa et al., 1996). AdoMetsynthetases from different sources are also capable of forming S-adenosyl-L-ethionine (AdoEt) or S-adenosyl-L-propionine (AdoProp) from ATP and L-ethionine or L-propionine, respectively, but largermethyl replacements are not well tolerated (Parks, 1958; Schlenk et al., 1975)..13

2.1 Synthesis of model AdoMet analogsH 2 NCO 2 HNH 2H 2 NCO 2 HNH 2HORCF 3 SO 2(CF 3 SO 2 ) 2 OOPVPCH 2 Cl 2SONNNNHCOOH:CH 3 COOH-CF 3 SO - 3S +ONNNNRHOOHRHOOHR=-H;-CH 3 ;-CH 2 -CH 3Figure 2. Chemical synthesis of model AdoMet analogs AdoPropin (R = -H), AdoButin (R = -CH 3 ) andAdoPentin (R = -CH 2 CH 3 ). Alcohol activation with triflic anhydride followed by AdoHcy alkylation.In order to overcome these restrictions, chemical synthesis of the desired AdoMet analogs wasattempted. AdoMet analogs were chemically synthesized by direct chemoselective alkylation ofAdoHcy under mild acidic conditions (a 1:1 mix of acetic and formic acids). No protecting groups areneeded because the acidic conditions lead to protonation and hence transient protection of allnucleophilic positions in AdoHcy except the sulfur atom (De La Haba et al., 1959).Trifluormethanesulfonic acid esters, prepared according to previously described procedure (Ross etal., 2000), were used as alkylating agents for the synthesis of the model AdoMet analogs. Like for thechemical synthesis of AdoMet from methyl iodide and AdoHcy the AdoMet analogs are obtained as anapproximately 1:1 diastereomeric mixture at the sulfonium center. Since only the (S)-diastereoisomerof AdoMet functions as cofactor for MTases (Borchardt et al., 1976), the protocol also includes achromatography step leading to pure (S)-AdoPentin and an enrichment of (S)-AdoButin isomers.Fractions leading to a higher DNA MTase activity are assigned to cofactor analogs with (S)-configuration at the sulfonium center.Figure 3. ESI-MS analysis of AdoMet, AdoPropin, AdoButin and AdoPentin. A. Mass spectra. (“*” indicatessodium ion adducts.) B. Cofactor fragmentation pathways.14

Chemical structure of the first analog, AdoButin, was confirmed by electrospray ionization massspectrometry (ESI-MS), high resolution mass spectrometry (HRMS) and 1 H NMR measurements(Table III), subsequent homologs,(AdoPentin) were characterized by ESI-MS. Additional informationwas gained from the fragmentation of cofactor molecular ion in the mass detector (Fig. 3, A). TheMass to charge ratio (m/z) of all fragments was in complete agreement with theoretical values.Furthermore, the fragmentation patterns of AdoMet and its analogs were very similar indicating similarchemical nature of these compounds (Fig. 3, B).2.2 Synthesis of cofactor analogs bearing a primary amino groupAdoMet analogs with an extended propargylic side chain carrying a primary amino group couldbe used for a two step labeling procedure: following the methyltransferase catalyzed transfer of theextended moiety onto DNA, reporter groups can be attached via chemoligation reaction with a varietyof amino-reactive probes in the second step (Kessler, 1994). Three such cofactors were synthesized:AdoButinNH 2 , AdoButinGABANH 2 and AdoHeksinGABANH 2 . Initial evaluation of AdoButinNH 2 andAdoButinGABANH 2 showed their rapid chemical conversion to corresponding hydrated species underthe enzymatic reaction conditions. Since this was not the case with AdoButin and AdoPentin, weassumed that the proximity of the amino and amido groups to the triple bond might be the reason forthe enhanced addition of a water molecule. This undesired effect could be overcome by isolatingamide group with additional –CH 2 - groups. Such assumption has led to the synthesis ofAdoHeksinGABNH 2 .The synthetic route to the amino analogs could be divided into three stages:a) N-Boc-protected aminoalcohol synthesis,b) N-Boc-protected aminoalcohol activation and coupling to AdoHcy,c) deprotection of reactive amino group in synthesized AdoMet analog.2.2.1 Synthesis of N-Boc-protected aminoalcoholsSynthesis of AdoButinNH 2 and AdoButinGABANH 2 was started from 2-butyn-1.4-diol, which wasconverted to 4-phthalimidobut-2-yn-1-ol according to modified previously described procedure (seematerials and methods section, Fig. 4, A). Afterwards it was converted into 4-amino-2-butyne-1-ol byhydrazinolysis. In case of AdoButinNH 2 , aminoalcohol was N-Boc-protected (Fig. 4, A) and activated(see next section). In case of AdoButinGABANH 2 , the hydrazinolysis product was extended bycarbodiimide (CDI) coupling with N-Boc-protected γ-aminobutyric acid (see materials and methodssection, Fig. 4, B).AOHOBHOOHNHOPh 3 P; DIAD56%NH 3++HOOHONHOONOO1. N 2 H 22. EtOH, HCl82%CDI, NEt 352%HOHONHNH 3+OBoc 2 O, NEt 3Figure 4. A. Synthesis of 4-(tert.-Butoxycarbonylamino)but-2-yn-1-ol. B. Synthesis of4-[(tert.-Butoxycarbonylamino)butanamido]but-2-yn-1-ol. Yields are indicated under the arrows.90%NHOOHOHNOOAminoalcohol for the synthesis of AdoHeksinGABANH 2 was obtained from 5-chloropent-1-yne,which was converted to 6-amino-2-hexyn-1-ol through coupling with formaldehyde followed by Gabrielsynthesis. The resulting product, similarly to previous synthesis, was extended by CDI coupling with N-Boc-protected γ-aminobutyric acid (see materials and methods section, Fig. 5).15

HOCl+HNH 3++OHHOBuLiTHFO70%NHHOOOCl1.CDI, NEt 360%ON - K + ,DMFO2. N 2 H 23. EtOH, HCl70%Figure 5. Synthesis of 6-[(tert.-Butoxycarbonylamino)butanamido]heks-2-yn-1-ol. Yields are indicated underthe arrows.All N-Boc-protected aminoalcohols were purified by column chromatography and the structureswere confirmed by 1 H and 13 C NMR (Table III).2.2.2 Activation of N-Boc-protected aminoalcohols, coupling to AdoHcy and N-Boc removalTriflate activation of N-protected aminoalcohols proved unsuitable due to a high reactivity of theinternal amide. A milder activator, 4-nitrophenylsulfonyl chloride, was found to be effective. Activationreaction was done according to modified previously described procedure (see materials and methodssection, Fig. 6, (Roberts, 1971)). The structure of obtained products was verified by 1 H and 13 C NMR(Table III).HOHONHONHONH 3+ONO 2HOOXONHO2NOSONaOHCH 2 Cl 240-60%ClOOOSONHOXH 2 NSCO 2 HNNOHO OHNH 2NN-HCOOH:CH 3 COOHOO 2 NS O -OOONHXH 2 NS +HOCO 2 HONNOHNH 2NNAdoButinNH 2 X=-CH 2 -AdoButinGABANH 2 X=-CH 2 -NH-C(O)-(CH 2 ) 3 -AdoHeksinGABANH 2 X=-(CH 2 ) 3 -NH-C(O)-(CH 2 ) 3 -Figure 6. Chemical synthesis of AdoMet analogs with amino group in the transferable chain. Protectedaminoalcohol was activated by 4-nitrobenzesulfonyl chloride and coupled to AdoHcy. Yields are indicated under thearrows.The activated N-protected aminoalcohols were directly coupled with AdoHcy under acidicconditions as described above (see materials and methods section). The Boc protecting group wasremoved by CF 3 COOH acid treatment (see materials and methods section, Fig. 7).H 2 NCO 2 HNH 2H 2 NCO 2 HNH 2NNNNS +NO XN 67%, CF 3 COOHONH-CO 2 ; - t-BuOHAdoHeksinGABANH 2 X=-(CH 2 ) 3 -NH-C(O)-(CH 2 ) 3 -~99%OHO OHAdoButinNH 2 X=-CH 2 -AdoButinGABANH 2 X=-CH 2 -NH-C(O)-(CH 2 ) 3 -Figure 7. Removal of Boc protecting group from cofactor analogs. Typical yield is indicated under the arrow.H 2 NXS +HOOOHNN162.3 Purification and structure elucidationIn contrast to model analogs, purification of amino-bearing cofactors was accomplished in severalstages (Fig. 8). 4-nitrobenzenesulfonic acid formed during alkylation reaction interferes with HPLC

purification and was therefore removed by passing the reaction mixture through an anion-exchangerDowex 1. Excess of CF 3 COOH, used for deprotection, was removed by filtering through reverse phaseC-18 silica gel. The final purification was carried out by reversed-phase HPLC (see materials andmethods section).Figure 8. Purification schemes of AdoMet analogs. The yields of individual diastereoisomers are indicated if theseparation by HPLC was achieved.Structure of the novel cofactors was verified by 1 H NMR and ESI-MS (see materials and methodssection). The ESI-MS fragmentation patterns were analogous with those observed for model AdoMetanalogs (see above and fig. 9). 1 H NMR data are presented in Table III.Figure 9. ESI-MS analysis of AdoButinNH 2 , AdoButinGABANH 2 and AdoHeksinGABANH 2 . A. Mass spectra. (“*”and “**” indicates sodium and potassium ion adducts respectively.) B. Cofactor fragmentation pathways.17

Table III. NMR spectral data of the synthesized compounds.Compound name1 H NMR signals δ, ppm13 C BMR signals δ, ppmProp-2-yn-1-ol triflate 5.00 (d, 4 J=2.5 Hz, 2H, CH 2 ) 2.77 (t, 4 J=2.5 Hz, 1H, CH) -But-2-yn-1-ol triflate 5.07 (k, 5 J=2.5 Hz, 2H, CH 2 ) 1.93 (t, 5 118.7 (k,J=2.4 Hz, 3H, CH 3 )C-F J =318 Hz; CF 3 ); 65.2; 89.6; 69.8;3.74-Aminobut-2-yn-1-ol3.77 (t, 5 J = 2.0 Hz, 2H, CHhydrochloride2 ), 4.18 (t, 5 J = 2.0, 2H, CH 2 ) 32.09; 52.23; 79.15; 87.964-(tert.-Butoxycarbonylamino)but- 1.45 (s, 9H, CH 3 ), 3.95 (s, 2H, CH 2 ), 3.95 (s, 2H, CH 2 );28.25; 30.50; 50.62; 80.08; 81.39; 81.55; 155.582-yn-1-ol5.08 (br. s., 1H, NH)1.47 (s, 9H, CH4-(tert.-Butoxycarbonylamino)but-3 ), 3.84 (s, 2H, CH 2 ), 4.61 (br. s., 1H, NH),28.55; 30.57; 59.34; 74.28; 80.71; 87.32;4.87 (s, 2H, CH2-ynyl-4-nitrobenzenesulfonate2 ); 8.09-8.26 (m, 2H, arom. H), 8.38-8.53124.67; 129.72; 142.36; 151.18; 171.87(m, 2H, arom. H)4-[(tert.-Butoxycarbonylamino)butanamido]but-2-yn-1-ol4-[4-(tert.-Butoxycarbonylamino)butanamido]but-2-ynyl-4-nitrobenzenesulfonate6-Chlorohex-2-yn-1-ol6-Aminohex-2-yn-1-olhydrochloride6-[(tert.-Butoxycarbonylamino)butanamido]hex-2-yn-1-ol6-[4-(tert.-Butoxycarbonylamino)butanamido]hex-2-ynyl-4-nitrobenzenesulfonateAdoButinAdoButinNH 2AdoButinGABANH 21.41 (s, 9H, CH 3 ), 1.78 (quint, 3 J = 6.8 Hz, 2H, CH 2 ), 2.22(t, 3 J = 7.1 Hz, 2H, CH 2 ), 3.13 (q, 3 J = 6.4 Hz, 2H, CH 2 ),4.03-4.09 (m, 2H, CH 2 ), 4.09-4.14 (m, 2H, CH 2 ), 4.84 (br.s, 1H, NH), 6.67 (br. s, 1H, NH)1.43 (s, 9H, CH 3 ), 1.76 (quint, 3 J = 6.6 Hz, 2H, CH 2 ), 2.19(t, 3 J = 7.1 Hz, 2H, CH 2 ), 3.13 (q, 3 J = 6.4 Hz, 2H, CH 2 ),3.92 (dt, 5 J = 1.8 Hz, 3 J = 5.3 Hz, 2H, CH 2 ), 4.74 (br. s, 1H,NH), 4.83 (t, 5 J = 1.8 Hz, 2H, CH 2 ), 6.61 (br. s, 1H, NH),8.09-8.17 (m, 2H, arom. H), 8.38-8.45 (m, 2H, arom. H)1.95 (quint, 3 J = 6.6 Hz, 2H, CH 2 ), 2.41 (tt, 3 J = 6.7 Hz, 5 J =2.2 Hz, 2H, CH 2 ), 2.77 (br. s., 1H, OH), 3.64 (t, 3 J = 6.4 Hz,2H, CH 2 ), 4.23 (t, 5 J = 2.2 Hz, 2H, CH 2 )1.88 (quint, 3 J = 7.5 Hz, 2H, CH 2 ), 2.39 (tt, 3 J = 6.9 Hz, 5 J =2.2 Hz, 2H, CH 2 ), 3.13 (t, 3 J = 7.5 Hz, 2H, CH 2 ), 4.22 (t, 5 J= 2.2 Hz, 2H, CH 2 )1.45 (s, 9H, CH 3 ), 1.69–1.87 (m, 4H, CH 2 ), 3.16 (t, 3 J = 6.5Hz, 2H, CH 2 ), 3.39 (q, 3 J = 6.5, 2H, CH 2 ), 4.24 (t, 5 J = 2.2Hz, 2H, CH 2 ), 5.06 (br. s, 1H, NH), 6.81 (br. s, 1H, NH)1.37 (s, 9H, CH 3 ); 1.55 (quint, 3 J = 7.0 Hz, 2H, CH 2 ), 1.74(quint, 3 J = 6.8 Hz, 2H, CH 2 ), 2.09 (tt, 3 J = 7.1 Hz, 5 J = 2.2Hz, 2H, CH 2 ), 2.19 (t, 3 J = 7.1 Hz, 2H, CH 2 ), 3.03-3.21 (m,4H, CH 2 ), 4.77 (t, 5 J = 2.2 Hz, 2H, CH 2 ), 5.13 (br. s., 1H,NH), 6.87 (br. s., 1H, NH), 8.07-8.13 (m, 2H, arom. H),8.33-8.40 (m, 2H, arom. H)1.70–1.73 (m, 0.9H, H4'' R ), 1.89 (t, 3 J = 2.1 Hz, 2.1H,H4'' S ), 2.32 (q, 3 J = 7.3 Hz, 2H, Hβ S/R ), 3.42–3.85 (m, 3.6H,Hγ S/R , Hα S/R , H5' R ), 3.93–4.00 (m, 1.4H, H5' S ), 4.18–4.23(m, 0.6H, H1'' R ), 4.27–4.32 (m, 1.4H, H1'' S ), 4.49–4.57 (m,1H, H4' S/R ), 4.63 (t, 3 J = 5.7 Hz, 1H, H3' S/R ), 4.92–4.97 (m,1H, H2' S/R ), 6.07 (d, 3 J = 4.2 Hz, 0.7H, H1' S ), 6.11 (d, 3 J =2.9 Hz, 0.3H, H1' R ), 8.25 (s, 1H, arom. H S/R ), 8.27 (s, 0.7H,arom. H S ), 8.28 (s, 0.3H, arom. H R ).2.18 (m, 2H, Hβ), 3.56-3.71 (m, 7H, Hα, Hγ, H5’, H1’’),3.87-3.96 (m, 2H, H4’’), 4.53-4.59 (m, 1H, H4’), 4.64 (t, 3 J= 5.9 Hz, 1H, H3’), 4.92 (dd, 3 J = 3.5 Hz; 5 J = 1.7 Hz, 1H,H2’), 6.10 (d, 3 J = 3.5 Hz, 1H, H1’), 8.26 (br. s., 2H, aromH)1.93 (quint, 3 J = 7.6 Hz, H7''), 2.29-2.44 (m, 4H, H8'', Hβ),3.01 (t, 3 J = 7.7 Hz, 2H, H6''), 3.45-3.75 (m, 2H, Hγ), 3.83(t, 3 J = 6.3 Hz, 1H, Hα), 4.00 (d, 3 J = 5.9 Hz, 2H, H5'), 4.05(br. s, 2H, H4''), 4.41 (br. s, 2H, H1''), 4.53 (q, 3 J = 5.9 Hz,1H, H4'), 4.65 (t, 3 J = 5.7 Hz, 1H, H3'), 4.93 (dd, 3 J = 3.8,5.3 Hz, 1H, H2'), 5.09 (t, 3 J = 1.7 Hz, 1H, NH), 6.11 (d, 3 J= 4.2 Hz, 1H, H1'), 8.30-8.35 (m, 2H, arom. H).26.38; 28.64; 29.62; 33.52; 40.05; 50.74; 79.63;81.22; 81.86; 153.87; 171.5326.96; 28.64; 29.26; 33.37; 39.66; 59.33; 74.19;79.89; 87.08; 124.69; 129.74; 142.36; 151.20;162.76; 172.6416.38; 31.45; 43.92; 51.20; 79.69; 84.3415.49; 25.78; 38.80; 49.91; 79.58; 84.6216.74; 26.65; 28.21; 28.66; 33.89; 39.01; 40.14;51.12; 79.73; 80.08; 84.99; 159.93; 173.4116.48; 26.59; 27.95; 28.59; 33.57; 38.75; 39.98;60.11; 72.23; 79.48; 90.72; 124.65; 129.69;142.36; 151.04; 156.87; 173.451.49 (quint, 1H, X 10 ), 1.65 (quint, 3H, H5’’), 1.82-1.92 (m,6H, H10’’, X 5 ), 2.08 (q, 1.2H, X 9 ), 2.20-2.35 (m, 10H, Hβ,H9’’, H4’’, X 4 ), 2.50 (t, 1.5H, X 6 ), 2.93-3.00 (m, 5.6H,H11’’), 3.06 (t, 1H, X 11 ), 3.14 (t, 1H, H6’’ R ), 3.22 (t, 1H,AdoHeksinGABANH 2 * H6’’ S ), 3.42-3.64 (m, 2.5 H, H5’ R , Hγ), 3.75-3.80 (m, 1H,Hα R/S ), 3.93-3.94 (m, 0.5H, H5’ S ), 4.29 (br. s, 1H, H1’’ R ),4.32 (br.s, 1H, H1’’ S ), 4.48-4.55 (m, 1H, H4’), 4.62 (t, 1H,H3'), 4.68 (t, 1.8H, X 1 ), 4.87-4.92 (m, 1H, H2’), 6.03-6.06(m, 1H, H1’ R/S ) 8.20-8.23 (m, 2H, arom. H)Note. X indicated signals from 6-in AdoHeksinGABANH 2 NMR spectrum 6-(4-aminobutanamido)hex-2-yn-1-ol.---18

3 Chemical stability of the AdoMet analogsThe stability of the synthesized AdoMet analogs was evaluated under physiological conditionsand in particular in the methyltransferase reaction buffers (pH 7.4). Reaction products were analyzedusing electrospray ionization mass spectrometry coupled to a liquid chromatograph (Fig 10, B).AdoMet is known to decompose via two pathways, one resulting in homoserine lactone and5’-(methylthio)adenosine, the second yielding adenine and S-pentosylmethionine (Fig 10, A). All theAdoMet analogs, except AdoButinNH 2 and AdoButinGABANH 2 , follow the same decomposition routes,however 4-7 times more rapidly than the natural cofactor (Fig. 10, C and Table IV).Figure 10. Stability of AdoMet analogs. A. Proposed decomposition pathways for AdoMet, AdoButin, AdoPentinand AdoHeksinGABANH 2 B. Reaction of AdoPentin decomposition in M.HhaI buffer (50 mM Tris-HCl, pH 7.4, 0.5mM EDTA, 2 mM 2-mercaptoethanol, 10 mM NaCl, 0.2 mg/ml BSA) as monitored by HPLC. The formed products areidentified by UV and mass spectrometry. C. Kinetic analysis of cofactors decomposition.19

Table IV. Decomposition kinetics of AdoMet and its analogs in aqueous buffers (pH 7.4).Cofactorh -1kAdoX/AdoMetτ 1/2 , hAdoMet 0.041±0.001 1 16.9±0.4AdoButin 0.22±0.01 5 3.2±0.2AdoPentin 0.149±0.006 4 4.6±0.2AdoButinGABANH 2 6.2±0.7 152 0.11±0.01AdoHeksinGABANH 2 0.278±0.008 7 2.49±0.07Figure 11. Chemical inactivation of AdoButinNH 2 and AdoButinGABANH 2 in MTase buffer. A. Proposedinactivation pathway via water molecule addition to the highly activated triple bond. B. Monitoring AdoButinGABANH 2hydration reaction by HPLC. C. Mass spectra of the hydration products. “*” and “**” indicates complexes with sodiumand potassium ions, respectively.Unexpectedly, LC/MS analysis of products formed upon incubation of AdoButinNH 2 andAdoButinGABANH 2 in aqueous buffers (pH 7.4) revealed a novel reaction pathway (Fig 11, A). It wasfound that a more polar (Fig 11, B) compound with a molecular mass of +18 a.m.u. is formed (Fig 11,C) in both cases. Such product was never observed with AdoButin or AdoPentin under similarconditions. The enhanced reactivity of the AdoButinNH 2 and AdoButinGABANH 2 cofactors to additionof nucleophiles (presumably hydroxyl anion) is likely due to highly electrophylic character of the triplebond which is surrounded by adjacent electronegative groups (sulfonium and amine or amide) Thehydratation of AdoButinGABANH 2 was ~150 times faster than AdoMet decomposition (see Fig. 11, Band Table IV) and the resulting hydration product was found to be inactive in enzymatic transalkylationreactions (see below). Since the hydration is strongly pH dependent, AdoButinNH 2 andAdoButinGABANH 2 could be stored for a reasonable period of time at pH = 3.5.20

4 Activity of AdoMet analogs in enzymatic reactions4.1 Activity with wild type cytosine DNA methyltransferasesThe model AdoMet analogs were tested with wild type DNA MTases, namely the DNA cytosine-N4 MTases BcnIB (recognition sequence CCSGG, modified nucleotide is underlined) and BfiIC2(ACTGGG), and the DNA cytosine-5 MTases HhaI (GCGC) and Eco31I (GGTCTC). The M.HhaI iswell characterized and serves as model system for structural and mechanistic studies of DNAmethylation.Figure 12. A. Evaluation of AdoMet and its analogs activity with WT M.HhaI by monitoring phage λ DNAprotection against R.Hin6I hydrolysis. Modification reaction was carried out in the M.HhaI reaction buffer at 37°Cfor 4 h, [cofactor] = 300 µM, [DNA targets] = 0.5 µM, [MTase]:[DNA targets] = 1:2 (N-1) , N – line number. „0“– controlreaction without MTase. The disappearance of fragmented DNA bands in lane N indicates 2 (N-1) full enzymaticturnovers in 4 h (or 2 (N-3) turnovers per 1 h). B. Analysis of enzymatic transalkylation reactions by N4-cytosine(M.BfiIC2 and M.BcnIB) and C5-cytosine (M.HhaI and M.Eco31IC) DNA MTases. Modifiable base withinrecognition site is underlined. Rates were estimated form DNA protection experiments shown above (end-pointassay).Enzymatic activities of the DNA MTases with the model AdoMet analogs were investigated usinga DNA protection assay. This assay makes use of the fact that DNA MTase catalyzed modification of anucleobase within the recognition sequence of a restriction endonucleases can protect the DNAagainst fragmentation by the restriction enzyme, which is easily analyzed by agarose gelelectrophoresis (see materials and methods section, Fig. 12, A). By varying the amount of DNA MTaseone can assess the reactivity of AdoMet analogs. The results of such analyses are summarized inFig. 12, B. Despite potential decline from linearity when approaching full protection in this end-pointassay, the observed turnover numbers for the wild-type M.HhaI in the presence of AdoMet (128 h -1 )agree well with previously reported steady-state values (k cat 144 h -1 ) (Vilkaitis et al., 2001).21

Consistent with previous observations (Parks, 1958; Schlenk et al., 1975), no or almost notransfer of the ethyl or propyl group from S-adenosyl-L-ethionine (AdoEt) and S-adenosyl-L-propionine(AdoProp) was observed under tested reactions conditions (Fig. 12, B), suggesting that enzymatictransfer of larger saturated alkyl chains would be hardly possible. Remarkably, the AdoMet analogswith methyl group replacements containing a carbon-carbon double (allylic system) or carbon-carbontriple bond (propargylic system) in β-position to the sulfonium center proved much more efficient.These cofactors rendered full modification of natural DNA at sub-stoichiometric amounts of DNAMTases over their recognition sites (Fig. 12) demonstrating that the poor reactivity of AdoMet analogswith saturated groups can be overcome by placing an activating group (unsaturated C-C bond) next tothe carbon unit that is attacked.Besides electronic factors, steric limitations imposed by the architectures of the catalytic sites inAdoMet-dependent enzymes may also affect the efficiency of enzymatic transalkylations by interferingwith cofactor binding or catalysis itself. The obtained results indicate that M.BcnIB possesses the mostopen active site which could accommodate the largest cofactor AdoPentin quite well producing 1turnover per hour (1 h -1 ). In contrast, M.BfiC2 showed almost no reactivity with AdoMet analogsindicating enzyme’s closed cofactor binding pocket. M.HhaI and M.Eco31I were capable of catalyzingbutynyl radical transfer, but struggle to use AdoPentin for DNA alkylation (Fig. 12, B). This indicatespartially obstructed cofactor binding site, which cloud be adapted to longer chain transfer bymutagenesis (see next section).4.2 Engineering the cofactor binding pocket in HhaI methyltransferaseInitial activity evaluation of the AdoMet analogs with transferable conjugated system showedstrongly reduced reactivity (30-100 times) compared to AdoMet. However, this contrasts with availableliterature data showing increased reactivity of allylic and propargylic systems (Dorwald, 2005). Toaddress inconsistency courses, MTase-DNA-AdoMet complex crystal structure of well characterizedDNA methyltransferase HhaI was examined (PDB id: 6mht) (Kumar et al., 1997). It was found thattransferable methyl group of AdoMet makes no contacts with protein residues in MTase-DNA-AdoMetcomplex. Based on this structure, MTase-DNA-AdoButin structure model was constructed. It showedthat Gln82 and Tyr254 residues might interfere with extended transferable side chain. Additionally,Asn304 residue could interact with bigger atoms replacing one of the methyl group hydrogens. Initialmutational investigation of these residues showed no significant changes in activity of M.HhaI(M. Čaikovskij, J. Šlyžiūtė, unpublished observations, (Lee et al., 2002)). Considering available data,the cofactor binding pocket of M.HhaI has been expanded by replacing Gln82, Tyr254 and Asn304with smaller amino acids (Ala or Ser).Figure 13. Models of the AdoMet (A) and AdoButin (B) bound in the active site of M.HhaI with DNA. MTase-DNA-AdoButin model was build based on MTase-DNA-AdoMet X-ray structure (PDB code 6mht). The arrow points tothe carbon unit that is enzymatically transferred to the C5 atom of the target cytosine residue. Pictures are made withSwiss-PdbViewer program (3.7 sp5 version) (Guex et al., 1997).22

All mutant enzymes demonstrated an increased activity towards model AdoMet analogs (Fig. 14).Gln82 change to Ala resulted in the biggest increase of enzymatic activity towards AdoPropen andapproximately 16 times decreased enzymatic activity towards natural cofactor. In contrast, Y254Smutation caused little effect on methylation activity, but significantly increased catalysis of triple bondbearing side chain transfer from AdoButin. The most dramatic activity changes arouse from N304Amutation. This variant was capable of catalyzing butynyl side chain transfer from AdoButin up to 8times more effectively than methyl from AdoMet. Asn304 of M.HhaI belongs to the conserved motif Xof cytosine C5-MTase family and is probably optimized for methyl group transfer. Our observationsindicate that smaller amino acid placed in this position might be more adequate for the catalysis ofextended side chain transfer (Fig. 14).Figure 14. AdoMet and its analogs activity with WT M.HhaI and its variants. Rates were estimated using theDNA protection assay (see fig. 12, A).Modeling of cofactor binding in the active site suggested that single mutations might not createenough space for large transferable groups (Fig. 13). To further optimize M.HhaI for the extended sidetransfer all possible enzymes bearing two and three mutations were constructed. Surprisingly, none ofdouble (Q82A/N304A or Y254S/N304A) or triple (Q82A/Y254S/N304A) mutant enzymes weresignificantly more efficient in transferring side chains from AdoButin or AdoPentin then M.HhaI N304Avariant (Fig. 13). This indicates that single mutation of Asn304 could create enough space in the activesite for the tested AdoMet analogs. Additionally, such hypothesis was supported by strongly reduced(8 and 32 times respectively) activity of Q82A/Y254S variant towards AdoButin and AdoPentincompared to M.HhaI N304A. Similarly Gln82 mutation seems to be more important for double bondbearing cofactor activity. Significant increase of catalysis efficiency towards AdoPropen was observedonly in Q82A/Y254S, Q82A/N304A and Q82A/Y254S/N304A variants (Fig. 13).23

Figure 15. AdoMet, AdoButinGABANH 2 and AdoHeksinGABANH 2 activity with WT M.HhaI and its variantsbearing N304A mutation. Rates were estimated by determining minimal amount of MTase required for λbacteriophage DNA protection from R.Hin6I hydrolysis (see fig. 12).Based on these results, enzymes possessing the N304A mutation were chosen for furtherinvestigation with cofactors bearing a reactive amino group. The best activities were shown by theY254S/N304A and Q82A/Y254S/N304A variants. In contrast, the N304A enzyme possessedsignificantly reduced activity towards these cofactors (Fig. 15) indicating that the single mutation is notsufficient for the catalysis of the extended side chain transfer from the cofactor.245 Composition analysis of transalkylated DNAThe DNA protection assay is convenient for initial testing of AdoMet analogs, but does not giveinformation about the chemical nature of a nucleotide formed during transalkylation reaction. Tocharacterize the nucleosides which are produced in the modified DNA, synthetic oligodeoxynucleotideduplex was incubated with the model cofactor analogs and Q82A M.HhaI or WT M.BcnIB. Thenucleotide composition of the product duplex was analyzed by enzymatic fragmentation to nucleosidesfollowed by reversed-phase HPLC (see material and methods section). In addition to the four naturalnucleosides, novel compounds with longer retention times were observed (Fig. 16, A). All newcompounds were identified by coupled ESI-MS as corresponding alkylated derivatives of 2′-deoxycytidine (Fig. 16, B and C).It should be noted that the molecular mass does not identify the position of the modification in anucleoside. This is of interest especially for modifications by M.HhaI and M.BcnIB which naturallymethylate cytosine residues at the C5 and N4 position, respectively. The following observationssupport the notion that the extended groups are transferred to the same position as observed for themethyl group: a) the extended groups are attached to the nucleobases because ESI-MS peakscorresponding to modified nucleobases are always detectable along with those of modifiednucleosides (Fig. 17, A); b) the chromatographic behavior of the modified nucleosides obtained withM.HhaI and M.BcnIB is different, indicating that different products are produced by the two DNAcytosine MTases (Fig. 17, B); c) UV spectra of all modified 2'-deoxycytidines obtained upon treatmentwith M.HhaI were similar to the UV spectrum of 5-methyl-2'-deoxycytidine, whereas, those modified by

M.BcnIB matched the UV spectrum of N4-methyl-2'-deoxycytidine (Fig. 17, C). In general, the UVmaxima of the expected C5 alkylation products occurred consistently at 6–10 nm higher wavelengthsas compared to the expected N4 alkylation products.Figure 16. Compositional analysis of products formed by M.HhaI Q82A in presence of AdoMet or modelcofactor analogs. A. HPLC chromatogram of nucleosides formed after enzymatic hydrolysis of transalkylated DNA.dA, dC, dG and dT stands for 2’-deoxyadenosine, 2’-deoxycytidine, 2’-deoxyguanosine and thymidine respectively. K– control experiment without cofactor. B. ESI-MS analysis of 2’-deoxycytidine and its modified derivatives. N denotesdeoxynucleoside; B - nucleobase. C. Fragmentation of nucleosides in the mass spectrometer.25

Figure 17. Properties of different alkylated derivatives of 2′-deoxycytidine formed in DNA by the action ofM.HhaI (Q82A variant) and WT M.BcnIB in presence of AdoPropen. A. ESI-MS spectra of the modifiednucleosides. N denotes deoxynucleoside; B - nucleobase. B. HPLC analysis of enzymatically fragmented duplexoligodeoxynucleotides (dR = 2'-deoxyribose) obtained after modification with M.BcnIB (red) or M.HhaI (Q82A variant,green) in the presence of AdoPropen (R = -CH 2 CH=CH 2 ). C. UV absorption spectra of modified 2'-deoxycytidines atpH 3.5 obtained with M.BcnIB and AdoMet (blue), M.BcnIB and AdoPropen (red), M.HhaI and AdoMet (orange) aswell as M.HhaI and AdoPropen (green).The stability tests of AdoButinNH 2 and AdoButinGABANH 2 indicated that these cofactors areprone to inactivating addition of a water molecule. Most probably, the water molecule is added to theactivating triple bond. To test if the hydration products can participate in the transfer reaction, DNAalkylated by Q82A/N304A M.HhaI or WT M.TaqI in the presence of AdoButinGABANH 2 orAdoHexinGABANH 2 was hydrolyzed to nucleosides and analyzed by LC\MS. It was shown that onlyproducts corresponding to unmodified side chains are formed (Fig. 18). This observation indicates thatAdoButinNH 2 and AdoButinGABANH 2 are inactivated by water molecule addition and hydratedderivatives do not participate in the enzymatic transalkylation reactions.26

Figure 18. Compositional analysis of products formed by M.HhaI Q82A/N304A in presence of AdoMet oramino-bearing cofactor analogs. A. HPLC chromatogram of nucleosides formed after enzymatic hydrolysis oftransalkylated DNA. dA, dC, dG and dT stands for 2’-deoxyadenosine, 2’-deoxycytidine, 2’-deoxyguanosine andthymidine respectively. K – control experiment without cofactor. B. ESI-MS analysis of 2’-deoxycytidine and itsmodified derivatives. N denotes deoxynucleoside; B - nucleobase. C. Fragmentation of modified cytosine base in themass spectrometer.6 Sequence specific blockage of protein-DNA interactionBesides methylation, other modifications are known to occur on natural DNA. For example, T-even bacteriophage genome DNA contains glycosylated hydroxymethylcytosine (Song et al., 1999)and β-D-glucosyl-hydroxymethyluracil is identified in the unicellular eukaryote Trypanosoma brucei (Yuet al., 2007). By modulating protein-DNA interactions the ‘very unusual’ (or hypermodified) basesserve various important biological functions, such as protection against degradation by nucleases andregulation of gene expression.Novel unusual bases are formed in the recognition sequences of DNA MTases duringmodification reactions in the presence of AdoMet analogs. It was hypothesized that deposited bulkygroups could interfere with the activity of restriction endonucleases by blocking protein-DNAinteraction. To test this hypothesis λ bacteriophage DNA was modified with M.HhaI or M.BcnIB in the27

presence of AdoPropen or AdoButin. Afterwards it was probed by digesting with R.BspLI, R.BglI andR.BseDI the recognition sequences of which partially overlap with M.HhaI and M.BcnIB targets (Fig.19). It was found that methylated λ bacteriophage DNA is hydrolyzed by tested restrictionendonucleases without interference, but butynyl or propenyl (only in case of M.BcnIB/R.BseDI pair)chain attachment is enough to inhibit cleavage of the modified DNA (Fig. 19).Figure 19. Blockage of restriction endonuclease cleavage at overlapping sites on λ DNA by enzymaticincorporation of extended groups. Phage lambda DNA was modified with M.HhaI or M.BcnIB and cofactorsAdoMet (R= -CH 3 ), AdoPropen (R= -CH 2 CH=CH 2 ) or AdoButin (R= -CH 2 C≡CCH 3 ) and then analyzed by cleavagewith the restriction endonucleases (targets indicated in brackets). Recognition sequences of MTases are indicatednearby the name, modifiable nucleotide is underlined. Altered fragmentation patterns (indicated by arrows) are due toblocked cleavage by the bulky propenyl or butynyl group at overlapping target sites.7 Targeted labeling of DNA by methyltransferase-directed Transfer of Activated Groups(mTAG)The utility of AdoButinGABANH 2 analog is readily demonstrated by labeling theamino-functionalized DNA with reporter groups using amine-reactive probes. For example, thepBR322 DNA premodified with M.HhaI variant Q82A/N304A or M.TaqI in the presence ofAdoButinGABANH 2 was further derivatized with a cyanine-5 fluorophore by treatment with acommercial NHS-ester (Fig. 20, A). Figure 20 (B) shows that the fluorescence intensity distribution infour pBR322-GsuI fragments is fully consistent with the positions and numbers of the M.HhaI andM.TaqI sites in the original plasmid (Table V). This observation confirms sequence specific andquantitative incorporation of the fluorophore.In addition, we used the mTAG technique for DNA methylation detection. pBR322 was treatedwith AdoButinGABANH 2 and M.TaqI and the amino-functionalized plasmid was labeled with NHSbiotin.In parallel, pBR322 was premethylated with the natural cofactor AdoMet and the BseCI DNAMTase (M.BseCI), then amino-functionalized with M.TaqI and treated with NHS-biotin. M.BseCImethylates the second adenine residue within the hexanucleotide 5'-ATCGAT-3' sequence, whichoverlaps with one of the seven M.TaqI sites in pBR322. Both plasmids were cleaved with the R.FspAIand R.MbiI endonucleases and the fragments passed over streptavidin beads to selectively capturebiotin-containing fragments (Fig. 21).28

Figure 20. Sequence-specific two-step labeling of plasmid DNA with cyanine-5 N-hydroxysuccinimide ester.A. Principal schema of two-step labeling procedure. B. Experimental results. pBR322 DNA was amino-modified withM.HhaI or M.TaqI in the presence of AdoButinGABANH 2 and then treated with a cyanine-5 N-hydroxysuccinimideester (Cy5-NHS). Labeled DNA was fragmented with R.GsuI endonuclease to produce fragments F1–F4 andanalyzed by agarose gel electrophoresis. Imaging of the Cy5 fluorescence was performed using a 635 nm laserscanner. A DNA fragment (F3) containing no M.TaqI targets is not visible in the scan. K - unmodified pBR322 DNAtreated with Cy5-NHS followed by R.GsuI fragmentation.Table V. Fluorescence intensity distribution in R.GsuI fragments of MTase-labeled pBR322 DNA.TaqI methyltransferaseHhaI methyltransferaseObserved peakObserved peakFragment TheoryTheoryarea, %area, %(size, bp)Number ofNumber of% Cy5 Fluoresceine% Cy5 Fluoresceinetarget sitestarget sitesF1 (1757) 4 57 60 55±7 14 45 43 42±2F2 (1434) 1 14 13 15±3 10 32 31 31±2F3 (616) 0 0 - - 4 13 14 15±2F4 (554) 2 29 28 31±10 3 10 12 12±329

Our experiment shows that quantitative M.TaqI-specific biotinylation is achieved. Mostimportantly, a different distribution pattern is observed with the M.BseCI-premethylated plasmid: theshortest fragment (F4′) is no longer retained on streptavidin-coated beads but is detected in the flowthroughfraction. Clearly, enzymatic methylation by M.BseCI blocks the single M.TaqI site on thisfragment for aminofunctionalization by M.TaqI and subsequent biotinylation, and therefore methylatedand unmethylated target sites can be quantitatively discriminated (Fig. 21). This result demonstratesthe utility of mTAG labeling to query the methylation status of specific sequences in DNA, paving theway to novel approaches for methylation profiling of genomic DNA.Figure 21. Sequence-specific two-step labeling of plasmid DNA with biotin-NHS ester. pBR322 DNA wasamino-modified with M.TaqI in the presence of AdoButinGABANH 2 and then treated with biotin-NHS. Labeled DNAwas fragmented with R.FspAI and R.MbiI to produce fragments F1′–F4′. Left panel: Analysis by agarose gelelectrophoresis of all DNA fragments (T= total), fragments not retained on streptavidin-coated magnetic beads (F=flow through), and fragments recovered from streptavidin-coated magnetic beads by extraction withphenol/chloroform (B= bound). Right panel: Same analysis except that pBR322 DNA premethylated with M.BseCIand AdoMet was used. The changed distribution of fragments shows that M.TaqI-specific biotinylation is blocked atthe premethylated M.BseCI site.30

8 DiscussionDNA methyltransferases (MTases) naturally catalyze the transfer of the activated methyl groupfrom S-adenosyl-L-methionine (AdoMet) to N- or C-nucleophiles in heterocyclic bases. In the presentwork, the enzymatic reaction has been redesigned to achieve the transfer of extended linear chains.The first step is to synthesize AdoMet analogs with extended transferable side chains (Klimasauskaset al., 2007). This first problem was solved by developing a general chemical approach toregioselective S-alkylation of AdoHcy leading to a variety of extended derivatives of AdoMet. However,the chemical extension of the transferable side chain in the cofactor is no guaranty for successfulenzymatic transfer of such groups, and two additional chemical problems need to be addressed.First, methyl transfer reactions are known to proceed by an S N 2 reaction mechanism, whichinvolves a hybridization change at the reacting carbon from sp 3 to sp 2 and back to sp 3 , with inversionof configuration. During the transition state, a p orbital is formed at the reacting carbon that interactswith both the attacking nucleophile and the leaving group. As a consequence, the bond anglesbetween the nucleophile as well as the leaving group and the three substituents at the reacting carbonare ~90°, which leads to destabilizing steric interactions (Dorwald, 2005). Thus, it is easy tounderstand that by going from the methyl group to the ethyl or propyl chains, the destabilizing stericeffect increases and leads to a reduced reaction rate. This has been confirmed by observation ofstrongly reduced enzymatic activities for AdoMet analogs with saturated extended transferable groups(ethyl or propyl) (Parks, 1958; Schlenk et al., 1975) and Figure 12. Second, the active sites of the DNAmethyltransferases have been evolutionary optimized to catalyze the methyl group transfer fromAdoMet. In general, the geometry and the size of the cofactor binding centers in MTases may not wellsuited to accommodate such extended AdoMet analogs, again leading to reduced rates of enzymatictransfer. These two major problems had to be solved for successful use of DNA methyltransferases asspecific DNA labeling tools.Figure 22. Proposed S N 2 reaction acceleration mechanism. A. Transition state for the enzymatic transfer of themethyl (upper plane) and allylic (lower plane) group respectively from AdoMet and AdoPropen to a nucleophile. S N 2reactions at saturated carbon atoms proceed via a hybridization change from sp 3 to sp 2 and back to sp 3 withinversion of configuration. During the transition state a p-orbital is formed which interacts with both the attackingnucleophile (Nu Ө ) and the leaving group AdoHcy. Conjugation (parallel alignment, shown by arrows) of the p-orbitalwith the neighboring anti-bonding π-orbital should lead to stabilization of the transition state. B. Proposed potentialenergy change during the enzymatic transfer of the methyl group (green line) and conjugated system (red line) fromAdoMet and cofactor analog respectively.Here we demonstrate, that the rate of the S N 2 alkylation reaction can be rescued by chemicallyplacing a carbon-carbon double bond (allylic system) or a carbon-carbon triple bond (propargylicsystem) in the extended side chain next to the reactive carbon (Fig. 22, A). The observed rateenhancement is universal for all three known classes of DNA MTases: cytosine-5, cytosine-N4 andadenine-N6. The chemical rationale for this reactivation derives from conjugation (parallel alignment)of the p-orbital formed in the transition state with the neighboring bonding or anti-bonding π-orbital31

(Fig. 22, A). This electronic effect leads to a stabilization of the transition state and, hence, to anacceleration of the reaction (Fig. 22, B). The new synthetic cofactors are termed double-activatedAdoMet analogs because the reactive carbon located between the sulfonium center and theunsaturated bond is activated for transfer by both adjacent groups.One downside of the double activation of the side chain is a somewhat reduced chemical stabilityof such cofactors in physiological aqueous buffers (see table IV). AdoButin and AdoPentin decayslightly faster, but follow the same decomposition routes as the natural cofactor. However, the firstcofactors carrying functional groups, AdoButinNH 2 and AdoButinGABANH 2 , turned out to be even lessstable. Both are rapidly inactivated by addition of a water molecule to the side chain at neutral pH. Themost probable mechanism of this pH-dependent hydration reaction is a hydroxyl ion attack on thetriple bond which is further activated by a negative inductive effect from the nearby amino or amidegroups. This hypothesis has been tested by the synthesis of AdoHexinGABANH 2 , in which the triplebond is well separated (by 3 carbon units) from the amido group. In full support to the assumption, nohydration reaction was observed in this case, resulting in an enhanced chemical stability of thecofactor analog.To address the second problem, rational engineering of the cofactor binding center in M.HhaIwas attempted. Computer modeling suggested that the side chains of Gln82, Tyr254 and Asn304 maylead to unfavorable steric interactions with cofactor analogs containing extended methyl groupreplacements. A simple engineering (shortening) of these residues led to a substantial improvement ofthe alkyltransferase efficiency. The highest effects were obtained upon replacement of highlyconserved residues (Gln82 and Asn304) in from universally sequence motifs IV and X. It is very likelythat similar mutations in other DNA C5-MTases, and likely other classes of AdoMet-dependentMTases, could lead to enhanced reactivity of the extended AdoMet analogs (Fig. 23).Figure 23. Amino acid sequence alignment of regions corresponding to IV and X conserved motifs ofprokaryotic, viral and eukaryotic cytosine-C5 MTases. Arrows indicate Gln82 and Asn304 positions of M.HhaI inIV and X conserved motifs. Four degrees of conservation in descending order: black background with white text, darkgray background with white text, gray background with black text and white background with black text.Altogether, these efforts have led to a successful redesign of the enzymatic DNA cytosine-5methyltransferase reaction into a DNA cytosine-5 alkyltransferase reaction. Indeed the N304A,Q82A/N304A, Y254S/N304A and Q82A/Y254S/N304A mutants of M.HhaI show higher turnover32

numbers with synthetic cofactors, such as AdoButin, as compared to the natural cofactor AdoMet.MTase-directed transalkylations of DNA with these synthetic cofactors occur in a highly specificmanner, with full retention of sequence-, base- and atom-specificity. High transalkylation rates (up to512 turnovers per h) indicate that sequence-specific derivatization of DNA is suitable for routinelaboratory use. This provides us with a novel powerful tool for sequence-specific derivatization of DNA.Three major types of DNA modification can be envisioned. First, bulky groups can be introduced intospecific loci on DNA to block intermolecular interactions in the cell. This approach has beendemonstrated in vitro by blocking the action of a restriction endonuclease at a unique target site onλ bacteriophage DNA. Second, it was demonstrated that novel synthetic AdoMet analogs bearing aprimary amino group in combination with DNA MTases can serve as versatile laboratory tool forsequence-specific functionalization and subsequent labeling of DNA (Fig. 20 and 21). The aminofunctionalizedDNA offers a wide diversity of reporters that can be attached via NHS ester chemistryincluding fluorophores and affinity probes. The synthetic scheme can be readily adapted for attachingabiotic functionalities, such as terminal alkyne, azide, ketone or maleimide, allowing a wide variety ofchemistries after MTase-catalyzed transfer. Third, reporter groups can be directly appended to thepropargylic side chains permitting single-step covalent labeling of DNA. An important advantage of thischemistry, compared to aziridine cofactor mimics (Pljevaljcic et al., 2004), is that linear chains can beused as linkers, which is expected to cause minimal steric perturbance to the labeled biomolecules.In addition to the described examples, several other DNA, RNA and protein MTases were activewith this class of synthetic AdoMet analogs (E. Weinhold, G. Vilkaitis, G. Urbanavičiūtė, unpublishedobservations). REBASE currently lists DNA MTases of over 200 different recognitions specificities,offering unprecedented experimental control over sequence-specific manipulation of DNA. This newclass of molecular tools envisions many potential applications ranging from probes for geneticscreening technologies to molecular building blocks in DNA-based nanobiotechnology. Owing to theuniversal nature of AdoMet-dependent methylation in biology, the double-activated AdoMet analogsare potentially applicable for labeling all three major classes of biopolymers (DNA, RNA, proteins)using the corresponding MTases. In our opinion, this will lead to the rapid development of the as yetuncovered biotechnological potential of AdoMet-dependent MTases.33

CONCLUSIONS1. S-Adenosyl-L-methionine (AdoMet) analogs with transferable extended propargylic side chainshave been obtained by direct chemoselective S-alkylation of S-adenosyl-L-homocysteine.2. AdoMet analogs bearing unsaturated bonds next to the reactive carbon are more active inenzymatic S N 2 reactions compared to analogs with fully saturated transferable radicals. Rapidcofactor inactivation by water molecule addition occurs if strongly electronegative group (amineor amide) is placed near the triple bond.3. All three known classes of DNA methyltransferases are capable of catalyzing extended grouptransfer from AdoMet analogs onto DNA yielding corresponding adenine-N6, cytosine-N4 andcytosine-C5 derivatives.4. The cofactor binding center of a DNA cytosine-5 methyltransferase, M.HhaI, can be engineeredfor enhanced transfer of the extended groups onto DNA by shortening side chains of the Gln82,Tyr254 and Asn304 residues.5. Synthetic AdoMet analogs carrying a primary aliphatic amino group in the extended side chaincan be used for methyltransferase-directed sequence-specific labeling of natural DNA.34

LIST OF PUBLICATIONS1. Dalhoff, C*., G. Lukinavičius*, S. Klimašauskas and E. Weinhold (2006). Direct transfer ofextended groups from synthetic cofactors by DNA methyltransferases. Nature Chemical Biology,2(1): 31-2.2. Dalhoff, C., G. Lukinavičius, S. Klimašauskas and E. Weinhold (2006). Synthesis of S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of DNA bymethyltransferases. Nature Protocols, 1(4): 1879-1886.3. Lukinavičius, G., V. Lapienė, Z. Staševskij, C. Dalhoff, E. Weinhold and S. Klimašauskas(2007). Targeted Labeling of DNA by Methyltransferase-Directed Transfer of Activated Groups(mTAG). Journal of American Chemical Society, 129(10): 2758-9.4. Weinhold, E., C. Dalhoff, S. Klimašauskas and G. Lukinavičius. New S-adenosyl-L-methionineanalogs with extended activated groups for transfer by methyltransferases. International patentapplication WO/2006/108678;* designated equal contributions35

ACKNOWLEDGEMENTSThe presented work was carried out at the Institute of Biotechnology and was in part supportedby grants form the Howard Hughes Medical Institute, the Volkswagen-Stiftung, the Bundesministeriumfur Bildung und Forschung (BMBF) and the Lithuanian State Science and Study foundation.I thank my supervisor Prof. S. Klimašauskas for scholarly guidance and valuable advice duringthis thesis work. I would like to thank E. Merkienė, G. Vilkaitis, D. Daujotytė, Z. Staševskij, M.Čaikovskij, G. Urbanavičiūtė and J. Šlyžiūtė for sharing their knowledge and generous help, as well asto all my colleagues at the Laboratory of Biological DNA Modification for their assistance, discussionsand moral support. I am grateful to my students M. Krupovič, F. Grygas, G. Urbanavičiūtė, V. Lapienėand A. Lapinaitė for their valuable experimental contributions to this work.I would like to express my appreciation to our collaborator Prof. E. Weinhold and his group(Institut für Organische Chemie der RWTH, Aachen, Germany) for WT M.TaqI, possibility to learnAdoMet analogs synthesis art, insightful discussions and hospitality during my stays in Aachen.Special thanks to Christian Dalhoff who has prepared some of AdoMet analogs.I am grateful to Marytė Krenevičienė for NMR measurements.I thank Fermentas UAB for providing enzymes and kits and access to laboratory equipment.I would like to thank my family for the encouragement and sustained help. Special thanks to mywife Rūta Gerasimaitė for scientific and not scientific help.CURRICULUM VITAEName:Gražvydas LukinavičiusAddress: Laboratory of Biological DNA Modification,Institute of Biotechnology,Graičiūno 8, Vilnius, LT-02241Phone: +370 5 260 21 18E-mail:grazvyds@ibt.ltEducation:2000 B.Sc., Biochemistry, Vilnius University;2002 M.Sc., Biochemistry, Vilnius University.Employment:Laboratory of Biological DNA Modification, Institute of Biotechnology:2006-present Junior scientist;2002-2006 Ph.D. student in Biochemistry;2000-2002 research assistant.36

REZIUMĖGyvas organizmas yra sudėtinga dinamiška sistema, susidedanti iš tarpusavyje sąveikaujančiųbiopolimerų ir jų metabolitų. Tokioje sistemoje dažniausiai tyrinėjamos viena ar kelios pasirinktosbiomolekulės, kurios pažymimos reporterinėmis grupėmis. Mažiausia artefaktinių rezultatų gavimotikimybė yra tuomet, kai maža signalą generuojanti molekulė prijungiama griežtai apibrėžtoje tiriamoobjekto vietoje. Pavyzdžiui, didelė DNR molekulė galėtų būti žymima tik specifinėse sekose.Šiuo metu sukurta nemažai specifinių DNR sekų vizualizacijos metodų, paremtų nekovalentinesąveika. Didžiausias jų trūkumas yra tai, kad grįžtama sąveika neleidžia griežtai susieti žymėsgeneruojamo signalo ir taikinio sekos, o dalis žymės visuomet lieka neprisijungusi ir didina nespecifinįsignalą (foną). Ieškant būdų kovalentiškai pažymėti gamtines DNR molekules specifinėse sekoseperspektyvu naudoti su DNR sąveikaujančius fermentus. Gamtoje plačiai paplitusi kovalentinė DNRmodifikacija – metilinimas. DNR metiltransferazės atpažįsta specifines DNR sekas ir katalizuoja metilogrupės pernešimą nuo kofaktoriaus S-adenozil-L-metionino (AdoMet) ant citozino arba adenino baziųtose sekose. Tokiu būdu greta įprasto genetinio kodo yra užrašoma papildoma epigetinė informacija irišplečiamos DNR informacinės galimybės. Nors ir būdama svarbi biologiniuose procesuose, metilogrupė tinka tik izotopiniam žymėjimui.Tiriant DNR metiltransferazių veikimo mechanizmą nustatyta, kad metilo grupės pernešimas yranukleofilinio pakeitimo (S N 2 tipo) reakcija, kurios metu aktyvuotą kofaktoriaus metilo grupę atakuojaazoto arba anglies nukleofilai, esantys DNR (Ho et al., 1991). Manoma, kad S N 2 reakcijos eigojesusiformuoja penkiakoordinuota tarpinė būsena, kurioje atakuojamas metilo grupės anglies atomaspereina iš sp 3 į sp 2 hibridizaciją ir susidaro p-orbitalė. Viena iš priežasčių, kodėl anksčiau tyrinėtiAdoMet analogai (Parks, 1958; Schlenk et al., 1975) yra mažiau aktyvūs yra tai, kad pakeitus vieną išpernešamos metilo grupės vandenilių didesne grupe (etilo ar propilo) atsiranda erdviniai trukdžiaiatakuojančiam nukleofilui priartėti ir susidaryti penkiakoordinuotai tarpinei būsenai.Šio darbo metu ištyrinėtas susintetintų AdoMet analogų aktyvumas DNR alkilinimo reakcijosekatalizuojamose visų trijų žinomų tipų metiltransferazių. Jis buvo ženkliai didesnis nei anksčiauaprašytų AdoEt ir AdoProp. Manoma šį efektą nulėmė tai, kad nauji AdoMet analogai sulfonio centroatžvilgiu β-padėtyje turi nesotų C-C ryšį, o S N 2 reakcijos tarpinė būsena yra stabilizuojamakonjugacijos tarp susidarančios p-orbitalės ir nesotaus ryšio π-orbitalių. Todėl pereinamos būsenosenergija yra žemesnė, pati reakciją pagreitėja.Kadangi gamtiniai fermentai yra evoliuciškai prisitaikę katalizuoti metilo grupės pernešimą nuoAdoMet, daugeliu atveju fermento aktyvumas su prailgintais AdoMet analogais nėra optimalus. Neišimtis yra ir DNR metiltransferazė HhaI. Tai nedidelis (~37 kDa), C5-citozino metiltransferazių šeimosfermentas, kurio katalitinis mechanizmas, sąveika su DNR ir kofaktoriumi yra gerai ištyrinėti (Wu et al.,1987; Cheng et al., 1993; Vilkaitis et al., 2001; Merkiene et al., 2005; Neely et al., 2005; Estabrook etal., 2006). Duomenų gausa ypač palengvino fermento aktyvaus centro modeliavimą ir pritaikymąkatalizuoti ilgesnių grupių pernešimą. Gauti rezultatai parodė, kad mutantinė metiltransferazė supraplėstu aktyviu centru ženkliai geriau katalizuoja prailgintos grandinės pernešimo ant DNR reakciją.Gln82 ir Asn304, kurių mutacijos padarė didžiausią įtaką fermento aktyvumui su AdoMet analogais,įeina į IV ir X konservatyvius motyvus. Taigi pasinaudojant palyginiu nesunku identifikuoti analogiškasaminorūgštis ir kitose citozino-C5 DNR metiltransferazėse. Tikėtina, kad analogiškos mutacijospagerins ir kitų citozino-C5 DNR metiltransferazių aktyvumą su AdoMet analogais.Išsprendus AdoMet analogų aktyvumo fermentinėse reakcijose problemą atsirado galimybėpanaudoti juos biopolimerų funkcionalizavimui ir žymėjimui. Pasinaudojant DNR metiltransferazėmisHhaI ir TaqI kryptingai įvesta reaktyvi amino grupė į 5‘-GCGC-3‘ ir 5‘-TCGA-3‘ sekas, esančiaspazmidinėje DNR. Vėliau prie amino grupės buvo prijungti fluorescuojantys dažai Cy5 ir fluoresceinasbei giminingumo žymė - biotinas.37

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